Liver disease is a common cause of rising morbidity and mortality in the United States: approximately 400,000 patients suffer from chronic liver disease. More than 25,000 patients die each year from complications of end- stage liver disease, including 1700 while awaiting liver transplant, indicating the need for a better understanding of disease pathogenesis and improved therapies. Clinical outcomes in patients with liver injury and risk for liver cancer are highly gender-dependent and not fully explained by external risk factors. These clinical observations strongly suggest an impact of sex hormones in liver disease pathogenesis. Using the zebrafish (Danio rerio) model, we have successfully elucidated regulatory roles for both nuclear hormone and G protein-coupled receptor signaling in liver development and regeneration, conserved across vertebrate species, and identified compounds to treat toxic liver injury. We have also discovered in important role for estrogen signaling in blood vessel and stem cell formation. Over the last funding period, we have defined two distinct roles for estrogen during development, inhibiting hepatic progenitor differentiation into hepatocytes via nuclear hormone receptor esr2, and promoting hepatocyte growth via G protein-coupled estrogen receptor gper1, demonstrating that estrogen is an important modifier of liver specification and growth. Our Preliminary Studies also demonstrate that estrogen affects hepatic regeneration and cancer formation, consistent with the published data on the gender differences observed after liver injury and with liver cancer incidence. Our long-term goal is to understand the molecular and cellular mechanisms by which estrogen and its downstream signals affect the liver. Our objective here is to characterize the functional implications of estrogen signaling on liver growth during development, regeneration, and carcinogenesis. Our central hypothesis is that estrogen acts on different cell populations via nuclear hormone receptors and the G protein-coupled estrogen receptor GPER1 to affect diverse aspects of hepatic differentiation and growth. This hypothesis is derived from our work over the previous funding period and the observed sexual dimorphisms in clinical outcome for liver regeneration and cancer. The rationale for our work is that a detailed understanding of the impact of estrogen on liver growth will explain observed gender differences and reveal new targets for liver cancer treatment. In Specific Aim 1, we seek to define the cellular and molecular mechanisms of estrogen signaling during liver formation and growth through chemical and genetic modification of estrogen signaling and extensive phenotypic, histological and functional analysis. In Specific Aim 2, we will investigate the role of estrogen and GPER1 acting via PI3K/AKT/MTOR in liver regeneration and cancer, using innovative models of liver injury and carcinogenesis. Our results will reveal novel therapeutic targets to amplify liver cell number in vitro, enhance liver regeneration, and prevent cancer formation or progression in patients with cirrhosis.